Mutant AOX1 promoters

Abstract

A mutant Pichia pastoris alcohol oxidase 1 (AOX1) promoter of the wild type Pichia pastoris AOX1 promoter (SEQ ID No. 1) comprising at least one mutation selected from the group consisting of: a) a transcription factor binding site (TFBS), b) nucleotides 170 to 235 (−784 to −719), nucleotides 170 to 191 (−784 to −763), nucleotides 192 to 213 (−762 to −741), nucleotides 192 to 210 (−762 to −744), nucleotides 207 to 209 (−747 to −745), nucleotides 214 to 235 (−740 to −719), nucleotides 304 to 350 (−650 to −604), nucleotides 364 to 393 (−590 to −561), nucleotides 434 to 508 (−520 to −446), nucleotides 509 to 551 (−445 to −403), nucleotides 552 to 560 (−402 to −394), nucleotides 585 to 617 (−369 to −337), nucleotides 621 to 660 (−333 to −294), nucleotides 625 to 683 (−329 to −271), nucleotides 736 to 741 (−218 to −213), nucleotides 737 to 738 (−217 to −216), nucleotides 726 to 755 (−228 to −199), nucleotides 784 to 800 (−170 to −154) or nucleotides 823 to 861 (−131 to −93) of Seq ID No. 1, and combinations thereof.

The invention claimed is: 
     
       1. An isolated mutant  Pichia pastoris  alcohol oxidase 1 (AOX1) promoter of a wild type  Pichia pastoris  AOX1 promoter comprising SEQ ID NO: 1 for expression under derepression conditions, wherein nucleotides 694 to 723 of SEQ ID NO: 1 are deleted. 
     
     
       2. The promoter according to  claim 1 , wherein the isolated mutant  Pichia pastoris  AOX1 promoter further comprises
 a mutation of a transcription factor binding site (TFBS) selected from the group consisting of Hap1, Hsf, Hap234, abaA, Stre, Rap1, Adr1, Mat1MC, and QA-1F, wherein the transcription factor binding site (TFBS) Hap1 comprises nucleotides 54 to 58 of SEQ ID NO: 1, Hsf comprises nucleotides 142 to 149 and 517 to 524 of SEQ ID NO: 1, Hap234 comprises nucleotides 196 to 200, 206 to 210 and 668 to 672 of SEQ ID NO: 1, abaA comprises nucleotides 219 to 224 of SEQ ID NO: 1, Stre comprises nucleotides 281 to 285 of SEQ ID NO:1, Rap1 comprises nucleotides 335 to 339 of SEQ ID NO: 1, Adr1 comprises nucleotides 371 to 377 of SEQ ID NO: 1, Mat1MC comprises nucleotides 683 to 687 of SEQ ID NO: 1, and QA-1F comprises nucleotides 747 to 761 of SEQ ID NO: 1, and/or 
 at least one mutation selected from the group consisting of a mutation within nucleotides 304 to 350 of SEQ ID NO: 1, a mutation within nucleotides 364 to 393 of SEQ ID NO: 1, a mutation within nucleotides 434 to 508 of SEQ ID NO: 1, a mutation within nucleotides 509 to 551 of SEQ ID NO: 1, a mutation within nucleotides 552 to 560 of SEQ ID NO: 1, a mutation within nucleotides 585 to 617 of SEQ ID NO:1, a mutation within nucleotides 621 to 660 of SEQ ID NO: 1, a mutation within nucleotides 625 to 683 of SEQ ID NO:1, a mutation within nucleotides 729 to 763 of SEQ ID NO:1, a mutation within nucleotides 736 to 741 of SEQ ID NO: 1, a mutation within nucleotides 737 to 738 of SEQ ID NO: 1, a mutation within nucleotides 726 to 755 of SEQ ID NO: 1, a mutation within nucleotides 784 to 800 of SEQ ID NO: 1 and a mutation within nucleotides 823 to 861 of SEQ ID NO: 1. 
 
     
     
       3. The promoter according to  claim 2 , wherein the mutation is a deletion, a substitution, an insertion, an inversion and/or a multiplication. 
     
     
       4. A nucleic acid molecule comprising
 at least one mutant  Pichia pastoris  alcohol oxidase 1 (AOX1) promoter according to  claim 1 ; and 
 at least one nucleic acid encoding a protein (peptide) or a functional nucleic acid, wherein said promoter and said nucleic acid are operably linked together forming a single- or multi-copy expression cassette. 
 
     
     
       5. A vector comprising a mutant  Pichia pastoris  alcohol oxidase 1 (AOX1) promoter according to  claim 1  or the nucleic acid molecule according to  claim 4 . 
     
     
       6. An isolated cell comprising:
 at least one isolated mutant  Pichia pastoris  alcohol oxidase 1 (AOX1) promoter according to  claim 1 ; 
 at least one nucleic acid molecule according to  claim 4 ; or 
 at least one vector according to  claim 5 . 
 
     
     
       7. The cell according to  claim 6 , wherein said cell is selected from the group consisting of a eukaryotic cell, selected from a yeast cell, a methylotrophic yeast cell, a  Candida  cell, a  Hansenula  cell, a  Pichia  cell, a  Toruplosis , and a  Pichia pastoris  cell. 
     
     
       8. A kit for the expression of a selected protein comprising
 i) the vector according to  claim 5 , and 
 ii) a cell capable to express said protein under the control of the AOX1 promoter of the vector. 
 
     
     
       9. The kit according to  claim 8 , wherein said cell is selected from the group consisting of a eukaryotic cell, selected from a yeast cell, a methylotrophic yeast cell, a  Candida  cell, a  Hansenula  cell, a  Pichia  cell, a  Toruplosis , and a  Pichia pastoris  cell. 
     
     
       10. A method of expressing a recombinant protein, peptide or functional nucleic acid in a cell comprising the following steps:
 providing the nucleic acid molecule according to  claim 4  or a vector comprising the nucleic acid molecule according to  claim 4 , 
 transforming said cell with said vector or said nucleic acid molecule, culturing the transformed cell in a suitable culture medium, 
 expressing said protein, peptide or functional nucleic acid and 
 isolating said expressed protein, peptide or functional nucleic acid. 
 
     
     
       11. The method according to  claim 10 , wherein said cell is selected from the group consisting of a eukaryotic cell, selected from a yeast cell, a methylotrophic yeast cell, a  Candida  cell, a  Hansenula  cell, a  Pichia  cell, a  Toruplosis , and a  Pichia pastoris  cell.